Enhanced purification of phosphorylated and nonphosphorylated biomolecules by apatite chromatography

ABSTRACT

Methods are disclosed for the use of apatite chromatography, particularly without reliance upon phosphate gradients, for fractionation or separation of phosphorylated and nonphosphorylated biomolecules. Integration of such methods into multi-step procedures, with other fractionation methods are additionally disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. provisional applications Ser.No. 61/011,513 filed Jan. 18, 2008; 61/062,663 filed Jan. 28, 2008;61/069,859 filed Mar. 19, 2008; 61/070,841 filed Mar. 27, 2008;61/135,787 filed Jul. 24, 2008; 61/189,467 filed Aug. 20, 2008, each ofwhich are expressly incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates in certain embodiments to methods for enhancingthe fractionation or purification of phosphorylated andnonphosphorylated biomolecules by apatite chromatography in the presenceof one or more of borate compounds, sulfate compounds, monocarboxylatecompounds, and/or in the presence of calcium compounds. In certainembodiments, the invention may permit more effective removal ofphosphorylated contaminants from nonphosphorylated products. In otherembodiments, the invention may permit more effective removal ofnonphosphorylated contaminants from phosphorylated products. In these orother embodiments, the invention may improve pH control duringfractionation.

BACKGROUND OF THE INVENTION

Hydroxyapatite [HA] is a crystalline mineral of calcium phosphate with astructural formula of Ca₁₀(PO₄)₆(OH)₂. Fluorapatite may be prepared byfluoridating hydroxyapatite, creating a mineral with the structuralformula Ca₁₀(PO₄)₆F₂. Protein-reactive sites on both minerals includepairs of positively charged calcium ions (C-sites) and triplets ofnegatively charged phosphate groups (P-sites). C-sites interact withproteins via HA calcium chelation by protein carboxyl clusters. C-sitesinteract with phosphorylated solutes such as DNA, endotoxin,phosphoproteins, and lipid enveloped viruses via HA calcium coordinationby solute phosphate residues. Calcium chelation and coordination aresometimes referred to as calcium affinity. P-sites interact withproteins via phosphoryl cation exchange with positively charged proteinamino acid residues (Gorbunoff, Analytical Biochemistry 136 425 (1984);Kawasaki, J. Chromatography 152 361 (1985)). Hydroxyapatite is mostcommonly eluted with phosphate gradients. The strong calcium affinity ofphosphate suspends calcium chelation and coordination interactions,while its ionic character suspends phosphoryl cation exchangeinteractions. Some applications elute hydroxyapatite with combinationsof phosphate and chloride salts. Chlorides preferentially elute thephosphoryl cation exchange interaction while having relatively littleeffect on calcium affinity interactions. (Gagnon et al, BioprocessInternational, 4(2) 50 (2006)).

Native hydroxyapatite and fluorapatite can be converted tocalcium-derivatized forms by exposure to soluble calcium in the absenceof phosphate. (Gorbunoff, Anal. Biochem., 136 425 (1984)). This convertsP-sites into secondary C-sites, abolishing phosphoryl cation exchangeinteractions, increasing the number of C-sites, and fundamentallyaltering the selectivity of the apatite support. Small alkaline proteinstypified by lysozyme (13.7-14.7 Kda, pI 10.7) and ribonuclease (14.7kDa, pI 9.5-9.8) fail to bind to calcium-derivatized apatites, but mostother proteins bind so strongly that even 3 M calcium chloride isinadequate to achieve elution (Gorbunoff). Other chloride salts alsofail to achieve elution. Calcium-derivatized apatites are restored totheir native forms by exposure to phosphate buffer, at which point theymay be eluted by methods commonly applied for elution of native apatitesupports.

The effects of different salts on the selectivity of a given apatite areunpredictable. For example, in the absence of phosphate, sodium chlorideis unable to elute most IgG monoclonal antibodies from nativehydroxyapatite, even at concentrations in excess of 4 moles per liter(Gagnon et al, 2006, Bioprocess International, 4(2)50). This impliesextremely strong binding. In exclusively phosphate gradients, IgG istypically one of the latest eluting proteins, usually requiring 100-150mM phosphate. This also implies strong binding. When eluted with acombination of lower concentrations of both salts, such as 0.25 M sodiumchloride and 50 mM phosphate however, IgG is one of the earliest elutingproteins. Other paradoxes reinforce the point: increasing the sodiumchloride concentration in the presence of phosphate, which causes IgG tobind less strongly, has the opposite effect on DNA (Gagnon et al, 2005,Bioprocess International, 3(7) 52-55). Additionally, Bovine serumalbumin (BSA) elutes at about 100 mM phosphate without respect to sodiumchloride concentration; and lysozyme elutes at a higher phosphateconcentration than BSA in the absence of sodium chloride but fails tobind in the presence of 1 M sodium chloride.

Ammonium sulfate, sodium sulfate, and other sulfate salts are commonlyused for precipitation of proteins, or to cause proteins to bind tohydrophobic interaction chromatography media. They can also be used toenhance binding with biological affinity chromatography media such asprotein A, and have even been reported to cause proteins to bind to ionexchangers (Gagnon, 1996, Purification Tools for Monoclonal Antibodies,ISBN 0-9653515-9-9; Mevarech et al, 1976, Biochemistry, 15, 2383-2387;Leicht et al, 1981, Anal. Biochem., 114, 186-192; Arakawa et al, 2007,J. Biochem. Biophys. Met., 70, 493-498). Sulfates have occasionally beenreported for elution of ion exchangers at low concentrations forresearch applications but are seldom exploited in preparativeapplications due to concerns over protein precipitation (Kopaciewicz etal, 1983, J. Chromatogr., 266 3-21; Gooding et al, 1984, J. Chromatogr.,296, 321-328; Rounds et al, 1984, J. Chromatogr., 283 37-45). None ofthese methods is an appropriate model for apatites because none of themexploits calcium affinity for binding.

Several authors have concluded that, “The presence of . . . (NH₄)₂SO₄seems not to affect the elution [of hydroxyapatite].” (Karlsson et al,1989, in Protein Purification: Principles, High Resolution Methods, andApplications, Chapter 4, ISBN 0-89573-122-3). Even this referencementions the application of sulfate strictly in the context of phosphategradients. In the rare cases where alternatives to phosphate as aprimary eluting salt have been discussed in the literature, suggestionshave included calcium chloride, citrate and fluoride salts, but withoutmention of sulfates (Gagnon, 1996; Karlsson et al, 1989; Gorbunoff).Other publications indicate that sulfate salts in particular should beunsuitable as primary eluting agents for hydroxyapatite because “ . . .SO₃H do[es] not form complexes with calcium.” (Gorbunoff).

Borate salts have been likewise overlooked. Borate is occasionally usedin the field of chromatography as a buffering agent at pH values fromabout 8.8 to 9.8 (pK ˜9.24). It is also used infrequently at alkaline pHto modify the charge characteristics of cis-diol compounds toselectively enhance their retention on anion exchangers. In contrast tophosphates, chlorides, and sulfates, all of which exhibit molarconductivities of about 90 mS/cm, a 1 M solution of borate at pH 7 has amolar conductivity of about 9 mS.

Acetates have been compared to chlorides for hydroxyapatite separationof IgG from aggregates and were found to support inferior fractionation(Gagnon et al, Practical issues in the industrial use of hydroxyapatitefor purification of monoclonal antibodies, Poster, 22^(nd) nationalmeeting of the American Chemical Society, San Francisco, Sep. 10-14,2006 <http://www.validated.com/revalbio/pdffiles/ACS_CHT_(—)02.pdf>.Monocarboxylic acid salts have been otherwise neglected, and the elutionpotential of monocarboxylic zwitterions totally so.

Hydroxyapatite is used for purification of a wide variety ofbiomolecules, including proteins, phosphoproteins, carbohydrates,polynucleotides, and viral particles. The column is usually equilibratedand the sample applied in a buffer that contains a low concentration ofphosphate. Adsorbed biomolecules are usually eluted in an increasinggradient of phosphate salts. Alternatively, some biomolecules may beeluted in an increasing gradient of chloride salts, but both elutionformats impose disadvantages on purification procedures. The highphosphate concentration in which antibodies elute in phosphate gradientshas strong buffer capacity that may interfere with subsequentpurification steps. The high conductivity at which some biomoleculeselute in chloride gradients may also interfere with downstream steps.Both situations require either that the eluted biomolecule be dilutedextensively, or that it be buffer-exchanged, for example bydiafiltration, in order to modify the conditions to render thepreparation suitable for application to a subsequent purification step.Dilution and buffer exchange have a negative impact on processeconomics. As a result, apatite chromatography steps are often placed atthe end of a purification process. This tends to eliminate them fromconsideration as capture steps. It also discourages the use of HA as anintermediate step. A further disadvantage of chloride gradients is thatthe application of chloride to hydroxyapatite causes an uncontrolledreduction of pH. Acidic pH causes destruction of hydroxyapatite andrisks adverse affects to biomolecules bound to it.

Another limitation of hydroxyapatite with biomolecule purification isthat the binding capacity for some biomolecules is reduced at elevatedconductivity values. This strongly reduces its versatility since thesalt concentration of cell culture supernatants andbiomolecule-containing fractions from purification methods such as ionexchange and hydrophobic interaction chromatography, confers sufficientconductivity to reduce the binding capacity of hydroxyapatite to such anextent that it may not be useful for a particular application. Thisdisadvantage can be overcome by diafiltration or dilution of the sampleprior to its application to the hydroxyapatite column, but as notedabove, these operations increase the expense of the overall purificationprocess. Alternatively, the disadvantage can be ameliorated by using alarger volume of hydroxyapatite, but this increases process expense byrequiring larger columns and larger buffer volumes. It also causes theantibody to elute in a larger volume of buffer, which increases overallprocess time in the subsequent purification step.

SUMMARY OF THE INVENTION

The present invention in certain embodiments relates to methods offractionating or purifying a desired biomolecule from an impurepreparation by contacting said preparation with a native orcalcium-derivatized apatite chromatography support, then eluting thesupport in the presence of an ionic species which is a sulfate, borate,monocarboxylic organic acid salt or monocarboxylic zwitterion. Incertain embodiments the ionic species is the primary eluting ion in theeluent. In certain embodiments the eluent is substantially free ofphosphate as an eluting ion.

In certain embodiments of the inventions, a method for purifying abiomolecule from an impure preparation is provided wherein the impurepreparation is contacted with an apatite chromatography support ineither the calcium derivatized form or in its native form and theapatite support is converted to the other form prior to elution of thebiomolecule.

In certain embodiments of the invention, the desired biomolecule to befractionated or purified is a biomolecule other than an antibody orantibody fragment.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of some embodiments of the invention include thefollowing: 1) Calcium-derivatized apatites support higher bindingcapacity than native hydroxyapatite for most biomolecules, even at highconductivity values, thereby making apatite chromatography moreeffective as a capture method, or as an intermediate fractionation stepfollowing high-salt elution from another fractionation step such as ionexchange or hydrophobic interaction chromatography; 2)Calcium-derivatized apatites also produce unique selectivities that mayenable effective biomolecule fractionation, including removal ofaggregates, in situations where native apatites fail to do so; 3)biomolecules may be bound to a native apatite support which is thenconverted to the calcium-derivatized form to achieve a particularselectivity for elution or; 4) biomolecules may be bound to acalcium-derivatized apatite support which is them converted to thenative form for elution. 5) Sulfate, borate, and certain monocarboxylicacids or zwitterions are able to elute biomolecules from apatitesupports in the absence of phosphate; 6) Elution in the presence ofsulfate, borate, and certain monocarboxylic acids or zwitterionsproduces unique selectivities that permit effective fractionation ofbiomolecules that may not be adequately served by elution with phosphateor by combinations of phosphate and chloride; 7) Borate permits elutionof biomolecules at low conductivity values, and does so without imposingsignificant buffer capacity at neutral pH, thereby facilitating use ofthe eluted biomolecule in subsequent ion exchange chromatography stepswithout the necessity for intervening steps such as diafiltration; 8)Borate and certain monocarboxylic acids or zwitterions create anincrease in pH on contact with apatites which can be used to counteractthe effect of chlorides on pH, thereby attenuating or eliminating the pHreduction that otherwise accompanies the introduction of chlorides; 9)Sulfate differentially enhances the retention of phosphorylatedbiomolecules, thereby enhancing their separation from non-phosphorylatedbiomolecules.

In certain embodiments the ionic species is borate. In certainembodiments the borate is sodium borate or potassium borate. In certainsuch embodiments the primary eluting ion is borate. In certainembodiments the borate is present at a pH where the borate lackssubstantial buffering capacity; in certain such embodiments the pH isless than 8.7. In certain other embodiments the borate is present atgreater than 50 mM and at a pH where the borate has substantialbuffering capacity; in certain such embodiments the pH is 8.7 orgreater.

In certain embodiments the ionic species is sulfate. In certainembodiments the sulfate is sodium or potassium sulfate. In certainembodiments the sulfate is the primary eluting ion.

In certain embodiments the ionic species is a monocarboxylic acid salt.In certain such embodiments the monocarboxylate acid anion is formate,acetate, lactate, succinate, pyruvate, gluconate, glucuronate orproprionate. In certain embodiments the monocarboxylate is the primaryeluting ion.

In still other embodiments the ionic species is a monocarboxyliczwitterion. In certain such embodiments the monocarboxylate zwitterionis glycine, proline, lysine or histidine.

In some embodiments, the biomolecule preparation may be applied to theapatite chromatography support under conditions that permit binding ofthe desired biomolecule and contaminants, with purification beingachieved subsequently by application of an elution gradient. This modeof chromatography is often referred to as bind-elute mode.

In some embodiments, the impure biomolecule preparation may be appliedto the apatite chromatography support under conditions that preventbinding of the desired biomolecule, while binding contaminants. Thismode of application is often referred to as flow-though mode. Boundcontaminants may be removed subsequently from the column by means of acleaning step.

Suitable apatite chromatography supports include native hydroxyapatite,calcium-derivatized hydroxyapatite, native fluorapatite, andcalcium-derivatized fluorapatite.

In certain embodiments, elution may be achieved exclusively by means ofincreasing the concentration of the ionic species such as borate,sulfate, or monocarboxylic acids or zwitterions. In certain of suchembodiments such elution is achieved with a single ionic species as theeluting ion, e.g., borate or sulfate.

In some embodiments, elution may be achieved by borate in combinationwith calcium, magnesium, phosphate, sulfate, chloride, monocarboxylicacids or zwitterions, arginine, glycine, urea, or nonionic organicpolymers.

In some embodiments, elution may be achieved by sulfate in combinationwith calcium, magnesium, phosphate, borate, chloride, monocarboxylicacids or zwitterions, arginine, glycine, urea, or nonionic organicpolymers.

In some embodiments, elution may be achieved by monocarboxylic acids orzwitterions in combination with calcium, magnesium, phosphate, borate,sulfate, chloride, arginine, glycine, urea, or nonionic organicpolymers.

In certain embodiments, the method for purifying a biomolecule from animpure preparation containing said biomolecule includes the steps of (a)contacting the impure preparation with an apatite chromatographysupport, wherein the apatite chromatography support is in acalcium-derivatized form when it is contacted with the biomolecule and(b) substantially converting the calcium-derivatized apatitechromatography support to its native form prior to eluting thebiomolecule. In certain such embodiments the biomolecule is eluted withphosphate as the primary eluting ion.

In certain embodiments, the method for purifying a non-aggregatedbiomolecule from an impure preparation containing said biomoleculeinvolves the steps of (a) contacting the impure preparation with anapatite chromatography support, wherein the apatite chromatographysupport is in its native form when it is contacted with the biomoleculeand (b) substantially converting the native form apatite chromatographysupport to a calcium-derivatized form prior to eluting the biomolecule.In certain such embodiments the conversion of the apatite chromatographysupport to the calcium derivatized form causes elution of thebiomolecule of interest.

In certain embodiments, phosphorylated biomolecules of interest areseparated by a method of the invention from non-phosphorylatedbiomolecules. In other embodiments, non-phosphorylated biomolecules ofinterest are separated by a method of the invention from phosphorylatedbiomolecules.

Embodiments of the invention may be practiced in combination with one ormore other purification methods, including but not limited to sizeexclusion chromatography, protein A and other forms of affinitychromatography, anion exchange chromatography, cation exchangechromatography, hydrophobic interaction chromatography, mixed modechromatography, and various filtration methods. It is within the abilityof a person of ordinary skill in the art to develop appropriateconditions for these methods and integrate them with the inventionherein to achieve purification of a particular antibody or antibodyfragment.

Terms are defined so that the invention may be understood more readily.Additional definitions are set forth throughout this disclosure.

“Apatite chromatography support” refers to a mineral of calcium andphosphate in a physical form suitable for the performance ofchromatography. Examples include but are not limited to hydroxyapatiteand fluorapatite. This definition is understood to include both thenative and calcium-derivatized forms of an apatite chromatographysupport.

“Salt” refers to an aqueous-soluble ionic compound formed by thecombination of negatively charged anions and positively charged cations.The anion or cation may be of organic or inorganic origin. Anions oforganic origin include but are not limited to acetate, lactate, malate,and succinate. Anions of inorganic origin include but are not limited tochloride, borate, sulfate, and phosphate. Cations of organic origininclude but are not limited to arginine and lysine. Cations of inorganicorigin include but are not limited to sodium, potassium, calcium,magnesium, and iron.

“Borate” refers to ionic compounds of boron and oxygen such as, but notlimited to boric acid, sodium borate, and potassium borate.

“Phosphate” refers to salts based on phosphorus (V) oxoacids such as,but not limited to, sodium phosphate and potassium phosphate.

“Sulfate” refers to salts based on sulfur (VI) oxoacids such as, but notlimited to sodium sulfate and ammonium sulfate.

“Chloride” refers to salts such as, but not limited to sodium chlorideand potassium chloride.

“Monocarboxylic acid salt” or “Monocarboxylate” refers to organic acidsalts having a single carboxylic acid moiety including but not limitedto the sodium or potassium salts of formic, acetic, propionic, lactic,pyruvic, gluconic, or glucuronic acid.

“Monocarboxylic zwitterion” refers to a molecule containing a singlecarboxyl moiety and at least one moiety with a positive charge. Suitableexamples include but are not limited to the amino acids glycine,proline, lysine, and histidine.

“Nonionic organic polymer” refers to any uncharged linear or branchedpolymer of organic composition. Examples include, but are not limitedto, dextrans, starches, celluloses, polyvinylpyrrolidones, polypropyleneglycols, and polyethylene glycols of various molecular weights.Polyethylene glycol has a structural formula HO—(CH₂—CH₂—O)_(n)—H.Examples include, but are not limited to, compositions with an averagepolymer molecular weight ranging from 100 to 10,000 daltons. The averagemolecular weight of commercial PEG preparations is typically indicatedby a hyphenated suffix. For example, PEG-600 refers to a preparationwith an average molecular weight of about 600 daltons.

“Buffering compound” refers to a chemical compound employed for thepurpose of stabilizing the pH of an aqueous solution within a specifiedrange. Phosphate is one example of a buffering compound. Other commonexamples include but are not limited to compounds such as acetate,morpholinoethanesulfonic acid (MES), Tris-hydroxyaminomethane (Tris),and hydroxyethylpiperazinesulfonic acid (HEPES).

“Buffer” refers to an aqueous formulation comprising a bufferingcompound and other components required to establish a specified set ofconditions to mediate control of a chromatography support. The term“equilibration buffer” refers to a buffer formulated to create theinitial operating conditions for a chromatographic operation. “Washbuffer” refers to a buffer formulated to displace unbound contaminantsfrom a chromatography support. “Elution buffer” refers to a bufferformulated to displace the one or more biomolecules from thechromatography support.

“Biomolecule” refers to any molecule of biological origin, composite, orfragmentary form thereof.

“Phosphorylated biomolecule” refers to any biomolecule, composite orfragmentary form thereof that includes at least one phosphate residue.Phosphorylation may be natural or induced by chemical modification.Examples include but are not limited to nucleotides, polynucleotides,DNA, RNA, endotoxins, lipid enveloped virus, phosphoproteins,phosphopeptides, phosphorylated amino acids, lipoproteins (where thelipid moiety is phosphorylated), phospholipids, glycophosphates, andglycophospholipids.

“Nonphosphorylated biomolecule” refers to any biomolecule, composite orfragmentary form thereof that is devoid of phosphate residues. Examplesinclude but are not limited to proteins, peptides, amino acids, lipids,and carbohydrates. Examples of proteins include but are not limited toantibodies, enzymes, growth regulators, and clotting factors.

“Biomolecule preparation” refers to any composition containing abiomolecule which is desired to be fractionated from contaminants.

“Antibody” refers to any immunoglobulin or composite form thereof. Theterm may include, but is not limited to polyclonal or monoclonalantibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived fromhuman or other mammalian cell lines, including natural or geneticallymodified forms such as humanized, human, single-chain, chimeric,synthetic, recombinant, hybrid, mutated, grafted, and in vitro generatedantibodies. “Antibodies” may also include composite forms including butnot limited to fusion proteins containing an immunoglobulin moiety.

“Antibody fragment” refers to any antibody fragment such as Fab,F(ab′)₂, Fv, scFv, Fd, mAb, dAb or other compositions that retainantigen-binding function. Antibody fragments may be derived from humanor other mammalian cell lines, including natural or genetically modifiedforms such as humanized, human, chimeric, synthetic, recombinant,hybrid, mutated, grafted, and in vitro generated, from sources includingbut not limited to bacterial cell lines, insect cell lines, plant celllines, yeast cell lines, or cell lines of other origin. Antibodyfragments may also be derived by controlled lysis of purified antibodywith enzymes such as, but not limited to ficin, papain, or pepsin.

As it relates to the invention herein, the term “bind-elute mode” refersto an operational approach to chromatography in which the bufferconditions are established so that the desired biomolecule andcontaminants bind to the column upon application, with fractionationbeing achieved subsequently by modification of the buffer conditions.

As it relates to the invention herein, the term “flow-through mode”refers to an operational approach to chromatography in which the bufferconditions are established so that the desired biomolecule flows throughthe column upon application while contaminants are selectively retained,thus achieving their removal.

“Analytical application” refers to a situation in which the invention ispracticed for the purpose of identifying and or determining the quantityof the desired molecule in particular preparation, in order to obtaininformation pertinent to research, diagnosis, or therapy.

“Preparative application” refers to a situation in which the inventionis practiced for the purpose of purifying intact non-aggregated antibodyfor research, diagnostic, or therapeutic applications. Such applicationsmay be practiced at any scale, ranging from milligrams to kilograms ofantibody per batch.

Materials

1. Apatite Chromatography Support

Various apatite chromatography supports are available commercially, anyof which can be used in the practice of this invention. These includebut are not limited to hydroxyapatite and fluorapatite. “Ceramic”hydroxyapatite (CHT™) or “ceramic” fluorapatite (CFT™) refer to forms ofthe respective minerals in which nanocrystals are aggregated intoparticles and fused at high temperature to create stable ceramicmicrospheres suitable for chromatography applications. Commercialexamples of ceramic hydroxyapatite include, but are not limited to CHTType I and CHT Type II. Commercial examples of fluorapatite include, butare not limited to CFT Type II. Unless specified, CHT and CFT refer toroughly spherical particles of any diameter, including but not limitedto 10, 20, 40, and 80 micron. HA Ultrogel™ refers to a productcomprising microfragments of non-ceramic hydroxyapatite embedded inporous agarose microspheres.

The choice of hydroxyapatite or fluorapatite, the type, and averageparticle diameter suitable for a particular fractionation can bedetermined through experimentation by the skilled artisan.

The invention may be practiced in a packed bed column, afluidized/expanded bed column containing the hydroxyapatite orfluorapatite, and/or a batch operation where the hydroxyapatite orfluorapatite is mixed with the solution for a certain time.

Certain embodiments employ CHT or CFT packed in a column.

Certain embodiments employ CHT or CFT, packed in a column of about 5 mminternal diameter and a height of about 50 mm, for evaluating theeffects of various buffer conditions on the binding and elutioncharacteristics of a particular antibody preparation of antibodyfragment preparation.

Certain embodiments employ CHT or CFT, packed in columns of anydimensions required to support preparative applications. Column diametermay range from 1 cm to more than 1 meter, and column height may rangefrom 5 cm to more than 30 cm depending on the requirements of aparticular application.

Appropriate column dimensions can be determined by the skilled artisan.

2. Biomolecule Preparations

Biomolecule preparations to which the invention can be applied mayinclude unpurified or partially purified biomolecules from natural,synthetic, or recombinant sources. Unpurified preparations may come fromvarious sources including, but not limited to, plasma, serum, ascitesfluid, milk, plant extracts, bacterial lysates, yeast lysates, orconditioned cell culture media. Partially purified preparations may comefrom unpurified preparations that have been processed by at least onechromatography, precipitation, other fractionation step, or anycombination of the foregoing. The chromatography step or steps mayemploy any method, including but not limited to size exclusion,affinity, anion exchange, cation exchange, protein A affinity,hydrophobic interaction, immobilized metal affinity chromatography, ormixed-mode chromatography. The precipitation step or steps may includesalt or PEG precipitation, or precipitation with organic acids, organicbases, or other agents. Other fractionation steps may include but arenot limited to crystallization, liquid:liquid partitioning, or membranefiltration.

B. Description of the Method

In preparation for contacting the biomolecule preparation with theapatite support, it is usually necessary to equilibrate the chemicalenvironment inside the column. This is accomplished by flowing anequilibration buffer through the column to establish the appropriate pH,conductivity, concentration of salts; and/or the identity, molecularweight, and concentration of nonionic organic polymer.

The equilibration buffer for applications conducted in bind-elute modemay include phosphate salts at a concentration of about 5-50 mM, orcalcium salts at a concentration of about 2-5 mM, but not mixtures ofphosphate and calcium. It may optionally include a nonionic organicpolymer at a concentration of about 0.01-50%, and a buffering compoundto confer adequate pH control. Buffering compounds may include but arenot limited to MES, HEPES, BICINE, imidazole, and Tris. The pH of theequilibration buffer for hydroxyapatite may range from about pH 6.5 topH 9.0. The pH of the equilibration buffer for fluorapatite may rangefrom about pH 5.0 to 9.0.

In one embodiment, the equilibration buffer contains sodium phosphate ata concentration of about 5 mM at a pH of 6.7, in the presence or absenceof MES or Hepes at a concentration of about 20-50 mM.

In one embodiment, the equilibration buffer contains a calcium salt at aconcentration of about 2.5 mM, in the presence of Hepes at aconcentration of about 20-50 mM and a pH of about 7.0.

The biomolecule preparation may also be equilibrated to conditionscompatible with the column equilibration buffer before the invention ispracticed. This consists of adjusting the pH, concentration of salts,and other compounds.

After the column and biomolecule preparation have been equilibrated, thebiomolecule preparation may be contacted with the column. Saidpreparation may be applied at a linear flow velocity in the range of,but not limited to, about 50-600 cm/hr. Appropriate flow velocity can bedetermined by the skilled artisan.

In one embodiment of the bind-elute mode, a column equilibrated inphosphate to obtain a particular binding selectivity during columnloading may be switched to calcium to obtain a particular elutionselectivity. Or the opposite may be performed, with a columnequilibrated to calcium to obtain a particular binding selectivity, andthen switched to phosphate to obtain a particular elution selectivity.

In one embodiment of the flow-through mode, non-aggregated biomoleculeflows through the column and is collected, while aggregated biomoleculebinds to the column. The biomolecule preparation is followed with a washbuffer, usually of the same composition as the equilibration buffer.This displaces remaining non-aggregated biomolecule from the column sothat it can be collected. Retained aggregates may optionally be removedfrom the column with a cleaning buffer of about 500 mM sodium phosphate,among others.

In one embodiment of an application conducted in bind-elute mode, somecombination of unwanted biomolecules, intact non-aggregated biomolecule,and aggregated biomolecule bind to the column. The biomoleculepreparation is followed with a wash buffer, usually of the samecomposition as the equilibration buffer. This removes unretainedcontaminants from the column. Unwanted biomolecule fragments may beselectively displaced by a wash buffer that removes fragments withoutremoving intact non-aggregated biomolecule. Intact non-aggregatedbiomolecule is then eluted from the column under conditions that leaveaggregated biomolecule bound to the column. Retained aggregates mayoptionally be removed from the column with a cleaning buffer of about500 mM sodium phosphate, among others.

In one embodiment of the bind-elute mode, the wash buffer may have aformulation different than the equilibration buffer.

After use, the apatite column may optionally be cleaned, sanitized, andstored in an appropriate agent.

The invention may be practiced in combination with other purificationmethods to achieve the desired level of biomolecule purity. Theinvention may be practiced at any point in a sequence of 2 or morepurification methods.

C. EXAMPLES

Considerable variation in chromatographic behavior is encountered fromone biomolecule preparation to another. This includes variation in thecomposition and proportion of undesired biomolecule contaminants, intactbiomolecule, biomolecule fragments, and biomolecule aggregates, as wellas variation in the individual retention characteristics of the variousconstituents. This makes it necessary to customize the buffer conditionsto apply the invention to its best advantage in each situation. This mayinvolve adjustment of pH, the concentration salts, the concentration ofbuffering components, and the content of nonionic organic polymer.Appropriate levels for the various parameters and components can bedetermined systematically by a variety of approaches. The followingexamples are offered for illustrative purposes only.

Example 1 . Dynamic binding capacity comparison of native andcalcium-derivatized hydroxyapatite. A column of hydroxyapatite, CHT TypeII, 40 micron, 5 mm diameter, 50 mm height, was equilibrated at a linearflow rate of 300 cm/hr with 20 mM Hepes, 3 mM CaCl₂, pH 6.7. A sample ofprotein A purified IgG monoclonal antibody was applied to the column byin-line dilution at a proportion of 1 part antibody to 4 partsequilibration buffer. Dynamic breakthrough capacity at 5% breakthroughwas 114 mg/mL of hydroxyapatite. The experiment was repeated with anequilibration buffer of 20 mM Hepes, 3 mM CaCl₂, 1 M NaCl, pH 6.7.Dynamic capacity at 5% breakthrough was 43 mg/mL. The experiment wasrepeated with an equilibration buffer of 5 mM sodium phosphate, pH 6.7.Dynamic capacity at 5% breakthrough was 29 mg/mL. The experiment wasrepeated with an equilibration buffer of 5 mM sodium phosphate, 1 MNaCl, pH 6.7. Dynamic capacity at 5% breakthrough was 3 mg/mL. Thisexample illustrates the dramatic improvement in antibody bindingcapacity that is achieved by calcium derivatized apatite. It will berecognized by the skilled practitioner that a similar benefit may beobtained by substituting magnesium for calcium.

Example 2. Purification of a biomolecule from cell culture supernatanton native hydroxyapatite, eluted with a borate gradient. A column ofhydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mm height, wasequilibrated at a linear flow rate of 300 cm/hr with 5 mM sodiumphosphate, 20 mM Hepes, pH 7.0. A monoclonal antibody preparationconsisting of a mammalian cell culture supernatant previously filteredthrough a membrane with porosity of about 0.22 μm, and diafiltered toabout the same conditions as the equilibration buffer was applied to thecolumn. The column was eluted with a linear gradient to 1 M sodiumborate, 5 mM sodium phosphate, pH 7.0. The majority of contaminatingproteins eluted before the antibody. Non-aggregated antibody eluted atan average conductivity of about 5 mS/cm. Aggregates eluted later. Thecolumn was cleaned with 500 mM sodium phosphate, pH 7.0. It will berecognized by the person of ordinary skill in the art that elutedantibody may be further purified by additional purification methods, andthat the low conductivity and buffer capacity of the eluted antibodyfraction will facilitate such methods.

Example 3. Purification of an biomolecule from cell culture supernatanton native hydroxyapatite, eluted with a monocarboxylic acid (lactate)gradient. A column of hydroxyapatite, CHT Type I, 40 micron, 5 mmdiameter, 50 mm height, was equilibrated at a linear flow rate of 600cm/hr with 5 mM sodium phosphate, 20 mM Hepes, pH 7.0. 100 microlitersof a monoclonal antibody preparation consisting of a mammalian cellculture supernatant previously filtered through a membrane with porosityof about 0.22 μm, was injected onto the column and the column washedwith 2 column volumes of equilibration buffer. The column was elutedwith a 20 column volume linear gradient to 1 M sodium lactate, 20 mMHepes, pH 7.0. The majority of contaminating proteins eluted before theantibody and most of the remainder eluted later. Non-aggregated antibodyeluted at an average conductivity of about 20 mS/cm. Aggregates elutedlater. The column was cleaned with 500 mM sodium phosphate, pH 7.0.

Example 4. Purification of a biomolecule from cell culture supernatanton native hydroxyapatite, eluted with a borate gradient. The same columnwas prepared with the same buffers but with a different IgG monoclonalantibody. The majority of contaminating proteins eluted as previouslybut only about 70% of the antibody eluted within the gradient, with theremainder eluting with the aggregate in the 500 mM phosphate cleaningstep. The run was repeated but with 20 mM phosphate in the equilibrationand elution buffers. Under these conditions, more than 80% of theantibody eluted within the gradient with the remainder eluting with theaggregate in the 500 mM phosphate cleaning step. The run was repeatedbut with 30 mM phosphate in the equilibration and elution buffers. Underthese conditions, more than 90% of the antibody eluted within thegradient, while a small amount of antibody eluted with aggregates in the500 mM phosphate cleaning step. This example illustrates one way toadapt the procedure to antibodies that may not elute fully within thegradient in the absence of phosphate, or at low phosphateconcentrations. The phosphate concentration may be increased more ifnecessary. Alternatively or additionally, the borate concentrationand/or pH of the eluting buffer may be increased. It will be recognizedby the skilled practitioner that the low conductivity and bufferingcapacity of the borate-eluted product make it better suited forsubsequent purification by cation exchange chromatography than elutionin a sodium chloride gradient. It will be equally recognized that thesubstitution of borate with monocarboxylic acids or zwitterions withmolar conductivities lower than sodium chloride may confer a similarbenefit.

Example 5. Purification of a biomolecule from cell culture supernatanton calcium derivatized hydroxyapatite, eluted with a borate gradient. Acolumn of hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mmheight, was equilibrated at a linear flow rate of 300 cm/hr with 2.5 mMcalcium chloride, 20 mM Hepes, pH 7.0. A monoclonal antibody preparationconsisting of cell culture supernatant previously filtered through amembrane with porosity of about 0.22 μm and diafiltered to about thesame conditions as the equilibration buffer was applied to the column.The column was eluted with a linear gradient to 1 M sodium borate, 2.5mM calcium chloride, 10% PEG-600, pH 7.0. The majority of contaminatingproteins eluted before the antibody. Antibody aggregate eluted afternon-aggregated antibody. The column was cleaned with 500 mM sodiumphosphate, pH 7.0. PEG is known to have the general effect of enhancingthe separation between fragments, intact antibody, and aggregates onhydroxyapatite. The skilled practitioner will recognize how to adjustthe PEG concentration to optimize the results.

Example 6. Biomolecule capture on calcium-derivatized hydroxyapatite andelution in a sulfate gradient. A column of hydroxyapatite, CHT Type II,40 micron, 5 mm diameter, 50 mm height, was equilibrated at a linearflow rate of 300 cm/hr with 20 mM Hepes, 3 mM CaCl₂, pH 6.7. Cellculture supernatant containing approximately 60 mg monoclonal IgG wasequilibrated to 5 mM calcium by addition of 1 M calcium chloride at aproportion of 0.5%, then filtered to 0.22 microns. The sample wasapplied to the column. No antibody was detected in the flow-through. Thecolumn was washed with equilibration buffer, then eluted with a 20column volume (CV) linear gradient to 20 mM Hepes, 3 mM CaCl₂, 0.5 Msodium sulfate, pH 6.7. The antibody eluted in a single peak at about0.25 M sodium sulfate.

Example 7. Biomolecule capture on calcium-derivatized hydroxyapatite,conversion to native hydroxyapatite, and elution in a phosphategradient. A column of hydroxyapatite, CHT Type II, 40 micron, 5 mmdiameter, 50 mm height, was equilibrated at a linear flow rate of 300cm/hr with 20 mM Hepes, 3 mM CaCl₂, pH 6.7. Cell culture supernatantcontaining monoclonal approximately 40 mg IgG was equilibrated to 5 mMcalcium by addition of 1 M calcium chloride at a proportion of 0.5%,then filtered to 0.22 microns. The sample was applied to the column. Noantibody was detected in the flow-through. The column was washed with 5mM sodium phosphate, 20 mM MES, pH 6.7, then eluted with a 20 CV lineargradient to 300 mM phosphate, pH 6.7. The antibody eluted in a singlepeak at about 165 mM sodium phosphate. This example illustrates the useof calcium-derivatized hydroxyapatite to obtain high binding capacity,followed by conversion to and elution from native hydroxyapatite.

Example 8. Intermediate purification of a biomolecule by binding in thepresence of calcium, conversion to native apatite, and elution in asodium chloride gradient. A column of hydroxyapatite, CHT Type II, 40micron, 5 mm diameter, 50 mm height, was equilibrated at a linear flowrate of 300 cm/hr with 20 mM Hepes, 3 mM CaCl₂, pH 6.7. Approximately 50mg of protein A purified monoclonal IgG was equilibrated to 5 mM calciumby addition of 1 M calcium chloride at a proportion of 0.5%, thenfiltered to 0.22 microns. The sample was applied to the column. Noantibody was detected in the flow-through. The column was washed with 20mM Hepes, 10 mM sodium phosphate, pH 6.7, then eluted with a 20 CVlinear gradient to 20 mM Hepes, 10 mM phosphate, 1 M sodium chloride, pH6.7. The antibody eluted in a single peak at 0.6 M sodium chloride,followed by a well-separated aggregate peak.

Example 9. Unwanted fragment and aggregate removal from a partiallypurified biomolecule on native hydroxyapatite, eluted with a borategradient. A column of hydroxyapatite, CHT Type I, 40 micron, 8 mmdiameter, 50 mm height, was equilibrated at a linear flow rate of 300cm/hr with 5 mM sodium phosphate, 20 mM Hepes, pH 7.0. A monoclonalantibody preparation previously purified by protein A affinitychromatography was applied to the column. The column was eluted with alinear gradient to 1 M sodium borate, 5 mM sodium phosphate, 20 mMHepes, pH 7.0. The majority of fragments eluted before the antibody.Antibody aggregates and other contaminating proteins eluted afternon-aggregated antibody. The column was cleaned with 500 mM sodiumphosphate, pH 7.0.

Example 10. Bind-elute mode, comparison of biomolecule elution inphosphate and sulfate gradients. A column of hydroxyapatite, CHT TypeII, 40 micron, 5 mm diameter, 50 mm height, was equilibrated at a linearflow rate of 200 cm/hr with 20 mM Hepes, 3 mM CaCl₂, pH 6.7. Cellsupernatant containing a monoclonal IgM antibody was applied to thecolumn. The column was eluted with a 20 CV linear gradient to 20 mMHepes, 3 mM CaCl₂, 1.0 M sodium sulfate, pH 6.7. The center of the IgMpeak eluted about 415 mM sodium sulfate. DNA eluted at 855 mM sulfateunder these conditions. IgM aggregates did not elute within the sulfategradient and were removed in a subsequent wash step with 500 mMphosphate. The experiment was repeated except that the column wasequilibrated with 10 mM sodium phosphate pH 6.7 and eluted with a 20 CVlinear gradient to 500 mM sodium phosphate, pH 6.7. The center of theIgM peak eluted at about 207 mM phosphate, essentially co-eluting withDNA as revealed by its elution at 205 mM phosphate. IgM aggregates wereonly partially eliminated. This example again illustrates the dramaticdifference of selectivity between sulfate and phosphate gradients,specifically and dramatically highlights how sulfate gradients are moreeffective for removal of DNA from IgM preparations, and specificallyillustrates the superior ability of sulfate gradients to eliminateaggregates. It will also be apparent to the skilled practitioner thatthese results illustrate the ability of the method to eliminatenonphosphorylated contaminants, such as proteins, from a preparation ofa phosphorylated biomolecule, such as DNA.

Example 11. Improved pH control by the application of borate. A columnof hydroxyapatite was equilibrated to 5 mM sodium phosphate, pH 7.0. Agradient step of 0.5 M sodium chloride, 5 mM sodium phosphate, pH 7.0was applied to the column. This caused the pH to drop to about pH 5.9.The column was re-equilibrated to 5 mM phosphate pH 7.0. A gradient stepof 0.5 mM sodium chloride, 5 mM sodium phosphate, 50 mM sodium borate,pH 7.0 was applied to the column. Column pH dropped only to pH 6.7. Itwill be understood by the skilled practitioner that the same approachcan be used to control pH in any situation where the introduction of aneluting agent causes an unacceptable reduction of pH, and that theborate concentration can be adjusted to achieve the desired degree of pHcontrol. Like borate, the application of lactate to an equilibratedapatite support causes an increase in pH, which can likewise beexploited to manage uncontrolled pH reduction caused by chlorides. Theskilled practitioner will recognize that other monocarboxylic acids orzwitterions may be substituted to produce a similar effect.

Example 12. Biomolecule fractionation with a borate gradient. A columnof hydroxyapatite, CHT Type I, 40 micron, 8 mm diameter, 50 mm height,was equilibrated at a linear flow rate of 300 cm/hr with 5 mM sodiumphosphate, 20 mM Hepes, pH 7.0. A Fab preparation from papain digestionof an IgG monoclonal antibody was applied to the column. The column waseluted with a linear gradient to 1 M sodium borate, 5 mM sodiumphosphate, 20 mM Hepes, pH 7.0. The majority of contaminating Fcfragments eluted before the Fab. Intact antibody eluted after the Fab.The column was cleaned with 500 mM sodium phosphate, pH 7.0.

Example 13. Flow-through purification of a biomolecule oncalcium-derivatized apatite. A column of hydroxyapatite, CHT Type I, 20micron, 5 mm diameter, 50 mm height, was equilibrated with 10 mM sodiumHepes, 2.5 mM calcium chloride, pH 7.0 at a linear flow rate of 300cm/hr. Calcium chloride was added to a Fab digest to a finalconcentration of 2.5 mM, then loaded onto the column. The Fab wasunretained and flowed through the column at a purity of about 95%.

Example 14. Biomolecule purification by application to native apatite,eluted by conversion to calcium-derivatized apatite. A column ofhydroxyapatite, CHT Type I, 20 micron, 5 mm diameter, 50 mm height, wasequilibrated with 5 mM sodium phosphate, 10 mM Hepes, pH 7.0 at a linearflow rate of 300 cm/hr. A Fab preparation was titrated to 5 mM phosphateand loaded onto the hydroxyapatite column. The Fab was retained andeluted with a step to 10 mM Hepes, 2.5 mM calcium chloride, pH 7.0.Purity was greater than 95%.

Example 15. Bind-elute mode, comparison of elution of a phosphorylatedbiomolecule in phosphate and sulfate gradients. A column ofhydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mm height, wasequilibrated at a linear flow rate of 300 cm/hr with 20 mM Hepes, 3 mMCaCl₂, pH 6.7. A sample of DNA isolated from salmon sperm was applied tothe column. The column was eluted with a 20 CV linear gradient to 20 mMHepes, 3 mM CaCl₂, 1.0 M sodium sulfate, pH 6.7. The center of the DNApeak eluted at about 855 mM sodium sulfate. The experiment was repeatedexcept that the column was equilibrated with 10 mM sodium phosphate pH6.7 and eluted with a 20 CV linear gradient to 500 mM sodium phosphate,pH 6.7. The center of the DNA peak eluted at about 205 mM sodiumphosphate. This example illustrates the dramatic difference betweenselectivity of sulfate and phosphate gradients. It will be apparent tothe skilled practitioner that these results also show the ability ofsulfate gradients to achieve more effective removal of DNA thanphosphate gradients.

Example 16. Bind-elute mode, comparison of elution of a phosphorylatedbiomolecule in phosphate and sulfate gradients. A column ofhydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mm height, wasequilibrated at a linear flow rate of 300 cm/hr with 20 mM Hepes, 3 mMCaCl₂, pH 6.7. A sample of endotoxin prepared by phenol extraction fromSalmonella enterica serotype typhimurium was applied to the column. Thecolumn was eluted with a 20 column volume (CV) linear gradient to 20 mMHepes, 3 mM CaCl₂, 1.0 M sodium sulfate, pH 6.7. A minor fraction ofendotoxin eluted early in the gradient, followed by a DNA contaminantpeak at 855 mM sodium sulfate. The majority of the endotoxin failed toelute and was removed from the column by cleaning it with 500 mM sodiumphosphate, pH 6.7. The experiment was repeated except that the columnwas equilibrated with 10 mM sodium phosphate pH 6.7 and eluted with a 20CV linear gradient to 500 mM sodium phosphate, pH 6.7. A minor fractionof the endotoxin, corresponding to the early eluting population in thesulfate gradient, failed to bind in phosphate and flowed through thecolumn immediately upon application. The center of the primary endotoxinpeak eluted at 85 mM sodium phosphate. This example illustrates thedramatic difference between selectivity of sulfate and phosphategradients in general, specifically illustrates the ability of sulfategradients to achieve unique separations among differentiallyphosphorylated biomolecules, and specifically illustrates that somephosphorylated biomolecules do not elute from at least some apatitechromatography supports in sulfate gradients conducted in the absence ofphosphate. It will be apparent to the skilled practitioner that theseresults also show the ability of sulfate gradients to achieve moreeffective removal of endotoxin than phosphate gradients.

Example 17. Enhanced fractionation of a phosphorylated protein from anonphosphorylated protein by differential enhancement of thephosphorylated protein in a sulfate gradient. A column ofhydroxyapatite, CHT Type I, 40 micron, 5 mm diameter, 50 mm height, wasequilibrated at a linear flow rate of 600 cm/hr with 20 mM Hepes, pH7.0. A purified monoclonal antibody (unphosphorylated) and a purifiedalpha-casein (polyphosphorylated) were applied, and the column waseluted in a 20 column volume linear gradient to 500 mM phosphate. Theantibody eluted at 156 mM phosphate. Alpha-casein eluted at 223 mMphosphate. The experiment was repeated, except eluting the column with a20 column volume linear gradient to 1 M ammonium sulfate, 20 mM Hepes,pH 7.0. The antibody eluted at 308 mM sulfate. Alpha-casein did notelute within the sulfate gradient. This shows that retention of theunphosphorylated protein was increased by about 97%, while the retentionof alpha-casein was increased by at least 350%.

It will be understood by the person of ordinary skill in the art how tooptimize and scale up the results from experiments such as thosedescribed in the above examples. It will also be understood by suchpersons that other approaches to method development, such as but notlimited to high-throughput robotic systems, can be employed to determinethe conditions that most effectively embody the invention for aparticular antibody.

D. Additional Optional Steps

The present invention may be combined with other purification methods toachieve higher levels of purification, if necessary. Examples include,but are not limited to, other methods commonly used for purification ofbiomolecules, such as size exclusion chromatography, protein A and otherforms of affinity chromatography, anion exchange chromatography, cationexchange chromatography, hydrophobic interaction chromatography,immobilized metal affinity chromatography, mixed mode chromatography,precipitation, crystallization, liquid:liquid partitioning, and variousfiltration methods. It is within the purview of one of ordinary skill inthe art to develop appropriate conditions for the various methods andintegrate them with the invention herein to achieve the necessarypurification of a particular antibody.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, chromatographyconditions, and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired performance sought to beobtained by the present invention.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

1. A method for purifying at least one non-aggregated biomolecule froman impure preparation containing said biomolecule comprising the stepsof (a) contacting the impure preparation with an apatite chromatographysupport and (b) conducting elution in the presence of an ionic speciesselected from the group consisting of borate, sulfate, monocarboxylates,and monocarboxylic zwitterions.
 2. The method of claim 1 wherein theionic species is borate or sulfate.
 3. The method of claim 1 wherein theionic species is a monocarboxylate selected from the group consisting ofacetate, proprionate, lactate, pyruvate, gluconate, and glucuronate. 4.The method of claim 3, wherein the monocarboxylate is sodium orpotassium lactate.
 5. The method of claim 1 wherein the ionic species isa monocarboxylic zwitterion selected from the group consisting ofglycine, proline, lysine, and histidine.
 6. The method of claim 1,wherein the monocarboxylic zwitterion possesses a pKa suitable forbuffering in the pH range selected for the particular purification, isused as the primary buffering species, and is present at a concentrationgreater than 50 mM.
 7. The method of claim 2, wherein the ionic speciesis borate and the borate is present at a pH where the borate lackssignificant buffer capacity.
 8. The method of claim 7, wherein the ionicspecies is borate and the borate is supplied as sodium borate orpotassium borate and is present at a pH of 8.7 or less.
 9. The method ofclaim 2, wherein the ionic species is borate and the borate is suppliedat a pH where the borate has significant buffer capacity, and the borateis present at a concentration greater than 50 mM.
 10. The method ofclaim 9, wherein the borate is sodium borate or potassium borate and ispresent at a pH of 8.7 or greater.
 11. The method of claim 2 wherein theionic species is borate and the borate is the primary eluting ion. 12.The method of claim 2 wherein the ionic species is sulfate and thesulfate is the primary eluting ion.
 13. The method of claim 3 whereinthe monocarboxylate is the primary eluting ion.
 14. The method of claim5 wherein the monocarboxylic zwitterion is the primary eluting ion. 15.The method of claim 1, wherein the apatite chromatography support ishydroxyapatite.
 16. The method of claim 1, wherein the apatitechromatography support is fluorapatite.
 17. The method of claim 1,wherein the apatite chromatography support is in its native form. 18.The method of claim 1, wherein the apatite chromatography support is ina calcium-derivatized form.
 19. The method of claim 1, wherein theapatite chromatography support is converted to its calcium derivatizedform after the step of contacting the impure preparation with theapatite chromatography support.
 20. The method of claim 1, wherein theapatite chromatography support is in equilibrium between its native formand a calcium-derivatized form.
 21. The method of claim 1, wherein theelution is conducted in the presence of one or more of an additionalsalt not comprising the ionic species, glycine, arginine, urea, or anonionic organic polymer.
 22. The method of claim 1, wherein thebiomolecule is not an antibody or immunoreactive antibody fragment. 23.The method of claim 1 wherein, the biomolecule fails to bind thecalcium-derivatized form of the apatite chromatography support.
 24. Themethod of claim 23, wherein the biomolecule of interest has a molecularweight greater than 15,000 daltons and an isoelectric point less than9.5.
 25. The method of claim 1 wherein the biomolecule is aphosphorylated biomolecule to be purified from non-phosphorylatedbiomolecules.
 26. The method of claim 25 wherein the biomolecule is apolynucleotide, vaccine, or lipid enveloped virus.
 27. The method ofclaim 26 wherein the biomolecule is a nucleic acid.
 28. A method forpurifying at least one non-aggregated biomolecule from an impurepreparation containing said biomolecule comprising the steps of (a)contacting the impure preparation with an apatite chromatographysupport, wherein the apatite chromatography support is in acalcium-derivatized form when it is contacted with the biomolecule and(b) substantially converting the calcium-derivatized apatitechromatography support to its native form prior to eluting thebiomolecule.
 29. The method of claim 28, wherein the converted nativeform chromatography apatite support is eluted with phosphate as theprimary eluting ion.
 30. The method of claim 29, wherein the biomoleculeis not an antibody or immunoreactive antibody fragment.
 31. A method forpurifying at least one non-aggregated biomolecule from an impurepreparation containing said biomolecule comprising the steps of (a)contacting the impure preparation with an apatite chromatographysupport, wherein the apatite chromatography support is in native formwhen it is contacted with the biomolecule and (b) substantiallyconverting the native form apatite chromatography support to acalcium-derivatized form prior to eluting the biomolecule.
 32. Themethod of claim 31, wherein conversion of the apatite chromatographysupport to the calcium derivatized form causes elution of thebiomolecule of interest.
 33. The method of claim 31, wherein thebiomolecule is not an antibody or immunoreactive antibody fragment.